AP® InvestIgAtIon #8 - apb.wikispaces.com Bio #8-1-35.pdf/518307418... · investigate factors that might alter the effectiveness of transformation. This lab ... pBR322, pUC18, pGLO

AP® InvestIgAtIon #8 TRANSFORMATION – TeAcheR’S GuIde

©2012, Ward’s Natural Science

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250-7457 v.5/12

Kit #36-7408

AbSTRAcT 1

GeNeRAl OveRvIew 2

RecORdING dATA IN A lAbORATORy NOTebOOk 3

MATeRIAl RequIReMeNTS/checklIST 5

cuRRIculuM AlIGNMeNT 7

leARNING ObjecTIveS 8

TIMe RequIReMeNTS 8

SAFeTy PRecAuTIONS 9

PRe-lAbORATORy PRePARATION 10

STudeNT GuIde cONTeNTS

bAckGROuNd 11

PART 1: bAcTeRIAl TRANSFORMATION wITh pucb 15

PART 2: TRANSFORMATION TeSTING vARIOuS PARAMeTeRS 20

ASSeSSMeNT queSTIONS 21

PART 3: bAcTeRIAl TRANSFORMATION 23

MATeRIAl SAFeTy dATA SheeTS 26

lIve cARe SheeTS 37

table of Contents

**AP® and the Advanced Placement Program are registered trademarks of the College Entrance Examination Board. The activity and materials in this kit were developed and prepared by WARD’S Natural Science Establishment, which bears sole responsibility for their contents..

note to sUMMeR InstItUte InstRUCtoRs

This kit was built with an error in the count of the sterile pipets and with some items that will not be used. We apologize and this issue will be addressed before shipping for classes this fall. For 8 student groups, we have included 16 sterile pipets instead of 100.

We have put instructions in capital letters for 8 groups to complete Part 1 with the enclosed materials.



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TRANSFORMATION: TeAcheR’S GuIde Kit #36-7408

AbstRACtStudents will transform common bacteria that are killed by antibiotics with a plasmid containing DNA that will confer antibiotic resistance. Students will calculate efficiencies of transformations and investigate factors that might alter the effectiveness of transformation. This lab illustrates the basic processes of genetic transformation, gene expression, and natural selection.



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geneRAl oveRvIewThe College Board has revised the AP Biology curriculum to begin implementation in the fall of 2012. Advanced Placement (AP) is a registered trademark of the College Entrance Examination Board. The revisions were designed to reduce the range of topics covered, to allow more depth of study and increased conceptual understanding for students. There is a shift in laboratory emphasis from instructor-designed demonstrations to student-designed investigations. While students may be introduced to concepts and methods as before, it is expected that they will develop more independent inquiry skills. Lab investigations now incorporate more student-questioning and experimental design. To accomplish this, the College Board has decreased the minimum number of required labs from 12 to 8 while keeping the same time requirement (25% of instruction time devoted to laboratory study). The College Board has defined seven science practices that students must learn to apply over the course of laboratory study. In brief, students must:

1. Use models

2. Use mathematics (quantitative skills)

3. Formulate questions

4. Plan and execute data collection strategies

5. Analyze and evaluate data

6. Explain results

7. Generalize data across domains

The College Board published 13 recommended laboratories in the spring of 2011. They can be found at: http://advancesinap.collegeboard.org/science/biology/lab

Many of these laboratories are extensions of those described in the 12 classic labs that the College Board has used in the past. The materials provided in this lab activity have been prepared by Ward’s to adapt to the specifications outlined in AP Biology Investigative Labs: An Inquiry-Based Approach (2012, The College Board). Ward’s has provided instructions and materials in the AP Biology Investigation series that complement the descriptions in this College Board publication. We recommend that all teachers review the College Board material as well as the instructions here to get the best understanding of what the learning goals are. Ward’s has structured each new AP investigation to have at least three parts: Structured, Guided, and Open Inquiry. Depending on a teacher’s syllabus, they may choose to do all or only parts of the investigations in scheduled lab periods.

The College Board requires that a syllabus describe how students communicate their experimental designs and results. It is up to the teacher to define how this requirement will be met. Having students keep a laboratory notebook is one straightforward way to do this.



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ReCoRdIng dAtA In A lAboRAtoRy notebookAll of the Ward’s Investigations assume that students will keep a laboratory notebook for student-directed investigations. A brief outline of recommended practices to set up a notebook, and one possible format, are provided below.

1. A composition book with bound pages is highly recommended. These can be found in most stationary stores. Ward’s offers several options with pre-numbered pages (for instance, item numbers 32-8040 and 15-8332). This prevents pages from being lost or mixed up over the course of an experiment.

2. The title page should contain, at the minimum, the student’s name. Pages should be numbered in succession.

3. After the title page, two to six pages should be reserved for a table of contents to be updated as experiments are done so they are easily found.

4. All entries should be made in permanent ink. Mistakes should be crossed out with a single line and should be initialed and dated. This clearly documents the actual sequence of events and methods of calculation. When in doubt, over-explain. In research labs, clear documentation may be required to audit and repeat results or obtain a patent.

5. It is good practice to permanently adhere a laboratory safety contract to the front cover of the notebook as a constant reminder to be safe.

6. It is professional lab practice to sign and date the bottom of every page. The instructor or lab partner can also sign and date as a witness to the veracity of the recording.

7. Any photos, data print-outs, or other type of documentation should be firmly adhered in the notebook. It is professional practice to draw a line from the notebook page over the inserted material to indicate that there has been no tampering with the records.

For student-directed investigations, it is expected that the student will provide an experimental plan for the teacher to approve before beginning any experiment. The general plan format follows that of writing a grant to fund a research project.

1. Define the question or testable hypothesis.

2. Describe the background information. Include previous experiments.

3. Describe the experimental design with controls, variables, and observations.

4. Describe the possible results and how they would be interpreted.

5. List the materials and methods to be used.

6. Note potential safety issues.

(continued on next page)



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ReCoRdIng dAtA In A lAboRAtoRy notebook (ContInUed)

After the plan is approved:

7. The step-by-step procedure should be documented in the lab notebook. This includes recording the calculations of concentrations, etc., as well as the weights and volumes used.

8. The results should be recorded (including drawings, photos, data print outs, etc.).

9. The analysis of results should be recorded.

10. Draw conclusions based on how the results compared to the predictions.

11. Limitations of the conclusions should be discussed, including thoughts about improving the experimental design, statistical significance, and uncontrolled variables.

12. Further study direction should be considered.

The College Board encourages peer review of student investigations through both formal and informal presentation with feedback and discussion. Assessment questions similar to those on the AP exam might resemble the following questions, which also might arise in peer review:

• Explain the purpose of a procedural step.

• Identify the independent variables and the dependent variables in an experiment.

• What results would you expect to see in the control group? The experimental group?

• How does XXXX concept account for YYYY findings?

• Describe a method to determine XXXX.



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MAteRIAls CheCklIst

MAteRIAls PRovIded In kItMAteRIAls needed bUt not PRovIded

Units per kit Description

£ 1 Disposable inoculating loop, ster. pkg/25 Microfuge tube racks

£ 4 Luria plates, pkg/10 Timer

£ 4 Luria plates w/ampicillin, pkg/10 Hot plate or temp-controlled water bath

£ 1Coupon for plasmid, pUC8 1 µg,

(100 ul) E. coli JM101*Thermometer

£ 1 Biohazard bag 1L flask

£ 30Centrifuge tubes, sterile, 120 mm, 15 mL

(extra component to be deleted; no current use)500 mL graduated cylinder

£ 16 Pipet, 6" sterile graduated, plastic Water

£ 1 Microfuge tubes, pkg/30, 1.5 mL Crushed ice in container

£ 6 Dilution tube, 125 mL × 16 mm,

filled with sterile broth10% solution of bleach

£ 1N,N-Dimethylformamide, 2 mL

(extra component to be deleted; no current use)Glass marking pens

£ 5 Calcium chloride, 10 mL tube Masking tape

£ 1 Sterile water, tube/4 mL Micropipettors with sterile tips 2–20 ul

£ 2 Disposable bacti-spreader, pkg/10 Ruler

£ 1 Instructions (this booklet) Gloves, safety goggles, lab aprons

Lab notebook

Heat protective gloves

Beakers

Additional materials as determined by students’ experimental designs


At least a week in advance of your lab, redeem the coupon provided for the perishable components, E. coli, pUC8 plasmid.

Store the vial of plasmid pUC8 frozen until needed.

Store the E. coli culture refrigerated: use or subculture within two weeks.

All other components may be stored at room temperature.

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oPtIonAl MAteRIAls (not PRovIded)

Incubator oven or seedling heat mat H2O

2 as a stress or a potential mutagen

UV light source Caffeine as a potential mutagen

Penicillin discs to look at antibiotic effects/resistance

Potassium nitrate

Streptomycin discs to look at antibiotic resistance

Luria agar, 200 mL bottle

Ampicillin, x-gal, IPTG to utilize the lac Z system

Other plasmids: pBR322, pUC18, pGLO, etc.

Capillary micropipets to substitute for sterile pipets

MAteRIAls CheCklIst (ContInUed)

Call “Us” at 1.800.962.2660 for

Technical Assistance Visit “Us” on-line at

www.wardsci.com

for U.S. Customers

www.wardsci.ca

for Canadian Customers

oR



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TRANSFORMATION: TeAcheR’S GuIde Kit #36-7408Kit # 3674-13

This lab activity is aligned with the 2012 AP Biology Curriculum (registered trademark of the College Board). Listed below are the aligned Content Areas (Big Ideas and Enduring Understandings), the Science Practices, and the Learning Objectives of the lab as described in AP Biology Investigative Labs: An Inquiry Approach (2012). This is a publication of the College Board that can be found at http://advancesinap.collegeboard.org/science/biology/lab.

CURRICUlUM AlIgnMentBig Ideas

Big Idea 3: Living systems store, retrieve, transmit, and respond to information essential to life processes.

And

Big Idea 1: The process of evolution drives the diversity and unity of life.

Enduring Understandings

1A2: Natural selection acts on phenotypic variations in populations.

1C3: Populations of organisms continue to evolve.

3A1: DNA, and in some cases RNA, is the primary source of heritable information.

3B1: Gene regulation results in differential gene expression.

3C1: Changes in genotype can result in changes in phenotype.

3C2: Biological systems have multiple processes that increase genetic variation.

Science Practices

3.1 The student can pose scientific questions.

5.3 The student can evaluate the evidence provided by data sets in relation to a particular scientific question.

6.4 The student can make claims and predictions about natural phenomena based on scientific theories and models.

7.1 The student can connect phenomena and models across spatial and temporal scales.

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leARnIng objeCtIvesThe student is able to connect evolutionary changes in a population over time to a change in the environment (1A2 & SP 7.1).

The student is able to evaluate given data sets that illustrate evolution as an ongoing process (1C3 & SP 5.3).

The student can justify the claim that humans can manipulate heritable information by identifying at least two commonly used technologies (3A1 & SP 6.4).

The student can predict how a change in a specific DNA or RNA sequence can result in changes in gene expression (3A1 & SP 6.4).

The student is able to pose questions about ethical, social, or medical issues surrounding human genetic disorders (an application of genetic engineering) (3A3 & SP 3.1).

tIMe ReqUIReMents

Part 1: Structured—Transformation Day 1 30–45 minutes to transform and plate

Day 2 15–45 minutes to collect and analyze data

Part 2: Guided—Transformation Day 1 30 minutes— could be same day as Part 1 Day 2

Optional— can be done concurrently with Part 1

Day 2 15 minutes

Part 3: Open Varies by student

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sAfety PReCAUtIons General Safety

The E. coli bacteria should be handled with care. Any items that come into contact with the culture should be treated with 20% bleach and disposed of in the biohazard waste. Biohazardous waste should be either autoclaved at 121 °C for 20 minutes or soaked in 20% bleach for 20 minutes. Check your school regulations before discarding in general trash or having biohazardous waste picked up for specialized disposal.

Ampicillin may cause allergic reactions so wear protective clothing at all times and wipe down bench tops and equipment daily. If contact occurs, rinse well with water and seek medical advice.

The teacher should 1) be familiar with safety practices and regulations in his/her school (district and state), and 2) know what needs to be treated as hazardous waste how to properly dispose of non-hazardous chemicals or biological material.

Consider establishing a safety contract that students and their parents must read and sign. This is a good way to identify students with allergies (i.e., latex) so that you (and they) will be reminded of specific lab materials that may be risks to individuals. A good practice is to include a copy of this contract in the student lab book (glued to the inside cover).

Students should know where all emergency equipment (safety shower, eyewash station, fire extinguisher, fire blanket, first aid kit, etc.) is located.

Make sure students remove all dangling jewelry and tie back long hair before they begin.

Remind students to read all instructions, MSDSs and live care sheets before starting the lab activities and to ask questions about safety and safe laboratory procedures.

In student directed investigations, make sure that collecting safety information (like MSDSs) is part of the experimental proposal.

As general laboratory practice, it is recommended that students wear proper protective equipment, such as gloves, safety goggles, and a lab apron to avoid staining any clothing or skin.

At the end of the lab:

All laboratory bench tops should be wiped down with a 20% bleach solution or disinfectant to ensure cleanliness.

Remind students to wash their hands thoroughly with soap and water before leaving the laboratory.

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PRe-lAboRAtoRy PRePARAtIonPrepare a starter plate of E. coli JM101 bacteria from the stock received through coupon redemption.

1. Subculture E. coli: The day before the experiment, obtain two (plain) Luria agar plates.

2. Using sterile technique and a sterile loop, streak bacteria on two plain Luria agar plates.

3. Invert the plates and place in a 37 °C incubator overnight. If an incubator is not available invert and allow to incubate at room temperature 24–48 hours. These plates will be used as starter plates.

4. Efficient transformation requires cells in an active growth phase.

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objeCtIves

Connect evolutionary changes in a population over time to a change in the environment.

Evaluate given data sets that illustrate evolution as an ongoing process.

Justify the claim that humans can manipulate heritable information by identifying at least two commonly used technologies.

Predict how a change in a specific DNA or RNA sequence can result in changes in gene expression.

Pose questions about ethical, social, or medical issues surrounding human genetic disorders (an application of genetic engineering).

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bACkgRoUndOne of the central ideas in biology is that the sequence of nucleotides in DNA determines the sequence in RNA and that sequence is translated into the amino acid sequence of the proteins. Changes in DNA will result in changes in protein expression or function thus changing the cells’ capabilities. Natural selection acts upon those changes in cellular function to change the composition of DNA in a population of organisms over time. Biotechnologists have developed a variety of tools to cause specific changes or additions to cellular DNA to cause changes in cellular function. In this laboratory, we will investigate one of those techniques, transformation, to change characteristics of the bacteria, E. coli.

Under natural circumstances, transformation involves the release of plasmid DNA from cells into the surrounding medium; subsequently recipient cells are able to take up the DNA from the medium. Plasmids are small circular molecules of double-stranded DNA, ranging in size from a few to several hundred kilobase pairs (Kbp) in length that function similarly to chromosomal DNA. Plasmids are self-replicating and can encode multiple proteins. However, unlike chromosomal DNA, they are not essential to the fundamental metabolic processes required for the growth and reproduction of the organism. The genes found on plasmids encode proteins involved in processes which benefit the cell in a limited set of environments: they can be thought of as accessory genes that aid in cell survival under specific conditions. Many, but not all, bacteria may contain anywhere from one to several dozen plasmids. Although they are capable of autonomous replication, the number of plasmids remains fairly constant from one generation of host cell to the next. Many plasmids, called R factors, carry genes that confer resistance to antibiotics upon the host cell. First discovered in 1955, R factors have spread rapidly among pathogenic bacteria in recent years, profoundly increasing public health risks by causing many strains of pathogenic bacteria to be highly resistant to antibiotics.

Most bacteria, like E. coli, do not take up plasmid DNA under normal conditions. In the laboratory, bacteria can be treated in a variety of ways that will permit them to take up plasmid DNA or become “competent” and able to be transformed. Artificial treatments that promote competence include electroporation and calcium chloride heat shock. In the heat shock procedure, DNA is dissolved in a solution of divalent cations (aqueous calcium chloride); the positive ions (Ca++) associate with the negatively charged DNA molecule to neutralize



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bACkgRoUnd (ContInUed)

notes it, rendering it less of a challenge to move across the selectively permeable cell membrane. The cells become competent only after the cells are suspended in ice cold calcium chloride solution containing the plasmid and subjected to shock by elevated temperature, causing the opening of pores of the bacterial cells thus allowing the plasmid DNA to enter the cells. The cells are then placed on ice to close their pores causing the plasmid to be retained. The presence of the foreign plasmid can be detected in the transformed cells through the detection of new characteristics (phenotypes) and altered genetics (genotype).

Artificial transformation has become one of the most important methodologies used in biotechnology. Today, many biotechnologists and pharmaceutical companies employ recombinant DNA technology and genetic engineering of bacteria to produce target proteins in high quantities (like human insulin) or to transform organisms like corn to be more resistant to disease.

Figure 1: The E. coli genome is more than 1000X larger than a plasmid.

E. Coli genome 4,639 kb

Plasmid 2.7 kb

Transformation System Properties

E. coli is a bacterial species commonly found in the human intestinal tract. It is available in many strains with different genetic backgrounds. We will be using the JM101 strain that is suitable for transformation with many plasmids.

The pUC8 is a small plasmid of about 2700 base-pairs, containing specific fragments of DNA that can confer antibiotic resistance to ampicillin in transformed wild type E. coli that are normally killed by ampicillin. Transformants of specific E. coli strains (like JM101) containing deletions of the lacZ gene and unable to metabolize lactose will survive ampicillin treatment and produce the alpha peptide of




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bACkgRoUnd (ContInUed)

notes B galactosidase due to the translation of genes in the plasmid DNA. When lactose positive cells are grown on media containing X-gal (a substrate for B-galactosidase that yields a blue precipitate) the transformed colonies will turn blue.

The pUC8 plasmid contains a polylinker site within the lacZ encoding sequence. The polylinker contains a variety of unique endonuclease restriction sites that permit the double stranded DNA to be cut at that particular site and no other in this plasmid causing the circular piece of DNA to become linear. Once the plasmid DNA is cut, defined pieces of DNA can be inserted into that site and re-attached to reform the circular plasmid from the linearized DNA with the new piece included in the polylinker site. This insertion has now disrupted the sequence of the lacZ gene so that transformants of lacZ negative bacteria strains with the new plasmid will show blue colonies for transformants without the insert and white colonies with the new insert under ampicillin selection.

Figure 2 is a map of pUC8. The total size of the plasmid is about 2700 base pairs. The ampicillin resistance gene is about 900 base pairs and contains several sequences that can be recognized by restriction endonucleases. The lacZ gene encodes a protein that can metabolize lactose. The polylinker site can be cut with a variety of restriction enzymes to permit the insertion of another gene. The polylinker is within the lacZ gene so that a successful insertion will disrupt the lacZ sequence and prevent functional B galactosidase from being made.

Figure 2

Ampicillin Resistance

Polylinker

lacZ

Origin of Replication

pUC8 Plasmid



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sAfety PReCAUtIons As general safe laboratory practice, it is recommended that students wear proper protective equipment, such as gloves, safety goggles, and a lab apron to avoid staining any clothing or skin. Ampicillin used in this laboratory may cause allergic reactions, so any area of contact should be rinsed immediately with water.

As general lab practice, read the lab through completely before starting, including Material Safety Data Sheets (MSDSs) and animal care sheets at the end of this booklet and find appropriate MSDSs for any additional substances the student would like to test. One of the best sources is the vendor for the material. For example, if purchased at Ward’s, searching for the chemical on the website will direct you to a link for the MSDS.

Sterile technique should be used throughout this laboratory to prevent contamination of experimental cultures. Instruments and solutions that came packaged as sterile should not be reused or come in unintentional contact with any non-sterile surface or non-sterile solution. If contamination occurs, discard and restart with sterile components. A sterile environment can be promoted by frequent wiping down of surfaces with 10% bleach.

At end of lab:

All laboratory bench tops should be wiped down with a 10% bleach solution or disinfectant to ensure cleanliness.

Students should wash their hands thoroughly with soap and water before leaving the laboratory.

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PARt 1 – stRUCtURed InqUIRy: bACteRIAl tRAnsfoRMAtIon wIth pUCb

MAteRIAls needed PeR lAb gRoUP1 Starter plate of E. coli JM1012 Luria agar plates2 Luria agar plates with ampicillin2 Microcentrifuge tubes2 Inoculating loops4 Bacti-spreaders4 Sterile graduated pipettes1 Water bath, 42 °C1 Water/ice bath, 0 °C2 Thermometers0.5 mL Calcium chloride0.5 mL Luria broth

PARt 1 – PRoCedURe: stRUCtURed InqUIRy1. Obtain two microcentrifuge tubes and mark one tube “+”, the

other “–”. The “+” tube will have the plasmid added to it; the “–” tube will act as a negative control.

2. OPEN STERILE PIPET PACKAGE PART WAY AND SLIDE THE PIPET OUT. Using a sterile graduated pipette, add 0.25 mL (250 ul) ice cold calcium chloride to each tube. Hold tubes on ice. For this measurement it is best to squeeze the stem of the pipette just a small amount, then insert the tip of the pipette into the liquid, then let up on the bulb and the liquid will be aspirated up to the first line on the stem of the pipette which is the 250 ul demarcation.

NOTE FOR INSTITUTES: PLEASE HOLD THIS PIPET AS STERILE FOR STEP 11 IF YOU ARE WORKING IN 8 GROUPS—INSTITUTE KITS WERE BUILT WITH AN ERROR IN COUNT. THIS WILL BE FIXED FOR FINAL KITS. TO HOLD AS STERILE, YOU MAY INSERT BACK INTO STERILE PACKAGING.

qqqqqqqqqqqq

PRoCedURe tIPs

When performing this lab activity, all data should be recorded in a lab notebook. You will need to construct your own data tables, where appropriate, in order to accurately capture the data from the investigation.

If directed to do so by your teacher, this part of the lab may be done at the same time as Part 2 of the lab.

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PARt 1 – PRoCedURe: stRUCtURed InqUIRy (ContInUed)

3. Obtain a starter plate of E. coli JM101. Use a sterile inoculating loop to transfer a large colony of bacteria from the starter plate to each tube of cold calcium chloride. Be sure not to transfer any agar to the tube.

4. To remove the bacteria from the transfer loop, and break them apart, place the loop into the calcium chloride and twirl rapidly. Dispose of the loop in the biohazardous waste as instructed.

TIP: Gently tapping the loop against the side of the tube may help dislodge the bacteria.

TIP: Prior to and after the period of heat shock be certain to keep the tubes of cells (and DNA) on ice.

5. You will use a sterile inoculating loop to transfer the plasmid solution to the “+” tube containing ice cold calcium chloride. Dip the loop into the DNA stock tube, when you remove the loop from the solution check to be sure that there is a drop of liquid contained in the loop area. Transfer the liquid (approximately 10 ul of the pUC8 plasmid solution, 10 ug of DNA per mL) to the microfuge tube labeled “+” only. Twirl loop to mix plasmid into solution.

6. Gently tap the tube with your finger to mix the plasmid into the solution.

7. Incubate both (“+” and “–”) tubes on ice for 15 minutes.

8. While the tubes are incubating, obtain two Luria agar plates and two Luria agar plates with ampicillin. Label one Luria agar plate “+”, the other “–”. Do the same for the Luria agar plates with ampicillin. Be sure to label all four plates with your group name.

TIP: Both time and temperature are critical in the following heat-shock steps. Be sure that your waterbath is at 42 °C and do not exceed 90 seconds in the waterbath. Cells must be on ice directly prior to, and immediately after the heat shock step.

9. Remove both of the tubes from ice and immediately place in a 42 °C waterbath for 60 seconds.

10. Remove the tubes from the 42 °C waterbath and immediately place on ice for two minutes.

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11. Remove the tubes from the ice bath and add 0.25 mL (250 ul) of room temperature Luria broth to each tube with a sterile disposable graduated pipette STARTING WITH THE “–” TUBE. Gently tap the tube with your finger to mix the solution. The tubes may now be kept at room temperature. DISPOSE OF THIS PIPET.

12. Add 0.1 mL (100 ul) of the “–” solution to the two “–” plates with a sterile disposable transfer pipette. For this measurement it is best to squeeze the stem of the pipette just below the bulb, then insert the tip of the pipette into the liquid, then let up on the stem and the liquid will be aspirated up to the second “bump” on the stem which is the 100 ul demarcation.

NOTE FOR INSTITUTES: PLEASE HOLD THIS PIPET AS STERILE FOR STEP 13 IF YOU ARE WORKING IN 8 GROUPS—INSTITUTE KITS WERE BUILT WITH AN ERROR IN COUNT. THIS WILL BE FIXED FOR FINAL KITS. TO HOLD AS STERILE, YOU MAY INSERT BACK INTO STERILE PACKAGING.

13. Add 0.1 mL (100 ul) of the “+” solution to the two “+” plates with a different sterile disposable graduated pipet. DISPOSE OF THIS PIPET.

14. Using a sterile Bacti-spreader, spread the cells over the entire surface of the Luria agar “–” plate. Then, using the same Bacti-spreader, spread the liquid on the Luria agar with ampicillin “–” plate.

15. Using a new sterile Bacti-spreader, repeat the previous procedure for both of the “+” plates. Spread the liquid on the Luria agar “+” plate first, followed by the Luria agar w/ampicillin “+” plate. Dispose of the Bacti-spreaders with biohazardous waste according to your instructor.

16. Let the plates sit for 5 minutes to absorb the liquid from the culture. Place the plates in a 37 °C incubator, inverted, overnight.

TIP: If an incubator is unavailable, the cultures within the petri dishes can incubate on the counter for 48–72 hours.

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notes 17. The next day, remove the plates from the incubator. Count and record the number of colonies on each plate.

TIP: When counting the colonies, DO NOT open the plate.

TIP: If there are too many colonies to count, divide the dish into quarters by marking the lid of the dish with a marking pen, count those colon in one quarter and multiply by four.

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PARt 1 – AnAlysIs of ResUltsDescribe the results observed on each plate:

Luria agar “+” _____________________________________________

Luria agar “–” _____________________________________________

Luria agar with ampicillin “+” ________________________________

Luria agar with ampicillin “–” ________________________________

A cell must be competent for transformation to occur. Not all cells in the solution become competent and therefore never receive the gene for antibiotic resistance. Transformation efficiency is the number of resistant colonies per microgram of plasmid. Using the directions below, calculate transformation efficiency.

Total mass of plasmid used (total mass = volume × concentration) __________________________

Total volume of suspension __________________________________

Fraction of cell suspension put on plate (µl on plate/total volume) ____________________________________

Total mass of plasmid in fraction (mass of plasmid × fraction on plate) ___________________________

Number of colonies per µg of plasmid (# of colonies counted/mass of plasmid put on plate) _______________

Sample Results: 25 colonies on Luria agar with ampicillin + plate

Total mass of plasmid used (total mass = volume × concentration) 10 μl × 1 μg/100 μl = 0.1 μg

Total volume of suspension 250 μl calcium chloride + 10 μl plasmid + 250 μl Luria broth = 510 μl

Fraction of cell suspension put on plate (µl on plate/total volume) 100 μl/510 μl = 0.196 or 20%

Total mass of plasmid in fraction (mass of plasmid × fraction on plate) 0.1 μg × 0.196 = 0.0196 μg

Number of colonies per µg of plasmid (# of colonies counted/mass of plasmid put on plate) 25/0.0196 or 1,276 colonies/μg

notes



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PARt 2 – gUIded InqUIRy: tRAnsfoRMAtIon testIng vARIoUs PARAMeteRsRepeat the experiment above to try to improve your transformation efficiency. You can vary the amount of time in heat shock, the temperature of heat shock, the number of bacterial colonies added to the transformation tube, the amount of plasmid, the concentration of CaCl, etc. Compare your transformation efficiency results to Part 1.

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PARt 2 – AssessMent qUestIons 1. Based on your experimental results in part 1, did transformation occur? Why or why not?

The “–” plates demonstrated growth on Luria agar, but no growth on Luria agar with ampicillin.

The “+” plates demonstrated growth on Luria agar and on Luria agar with ampicillin.

The cells in both the “+” tube and in the “–” tube were treated to induce competency; only the cells in the “+” tube were exposed to the plasmid pUC8. Therefore, the fact that only the cells from the “+” tube grew in the presence of ampicillin indicates that transformation did occur in the cells from that tube.

2. You repeated the experiment in Part 1 and examined the results the next day. Below is what your plates looked like when you checked them. Explain what may or may not have occurred.

Students should notice that no growth occurred on the Luria w/amp “+” plate. This means that transformation was not successful. Allow students to speculate and accept any reasonable answer. Examples of problems encountered may include killing the cells by heat-shocking them at too high of a temperature, the plasmid was not added to the cells to be transformed, there was something wrong with the plasmid, a reagent (such as CaCl

2) was omitted from the

transformation mixture, etc.

3. How would you interpret growth of colonies from non-plasmid treated cells on AMP+ plates?

This control would tell you that either the amp is not killing the bacteria (old plates with inactive amp?) or that the strain of bacteria used has some rate of cells that have spontaneously developed resistance to amp. Either way, you would want to repeat with fresh amp plates (most likely) and test another clone from your starter bacterial colony.

Bacterial Lawn Bacterial Lawn No Growth No Growth

Luria “–” Luria “+” Luria w/amp “–” Luria w/amp “+”



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4. If you did not have ampicillin to select for transformed colonies, given what you know about the pUC8 plasmid and the JM101 strain, is there another way you could determine transformation efficiency?

You could plate the cells on plates containing the B galactosidase substrate, XGAL, and count the number of blue colonies—these colonies would come from transformed cells. You might have to plate at a lower cell density to resolve colonies, but the calculation would be the same.

5. Were your efforts to improve the transformation efficiency successful? Why do you think you got the results you did? What else might you do to confirm your interpretation?

Answers will vary but here is a sampling:

Timeinheatshock:efficiencymaychange,butisitsignificant?Replicateexperimentswouldneedtobedonetoteststatisticalsignificance.Thereislikelyanoptimumtimesoexperimenting on a continuous range rather than one experimental point will give better information. Time too long likely to kill bacteria, but the experimental design does not permit you to see this. Appropriate control comparison would be no heat shock.

Tempofheatshock:efficiencymaychange,butisitsignificant?Samecommentsasabove.

Increased bacterial colonies: amp-control plates should have more colonies than the transformationinPart1,buttransformationefficiencymaynotincreaseifonecolonyalreadygives maximal transformation with that plasmid concentration. Additional experiments could bedonetodefineoptimalratiobetweenplasmidconcentrationandcolonyconcentrationunder these given transformation parameters.

Increased plasmid concentration: the number of transformed colonies per plate may increase,butoncenormalizedforplasmidconcentration,efficiencylikelytobethesame.Seeoptimization comments above to determine whether bacterial concentration is limiting.

PARt 2 – AssessMent qUestIons (ContInUed)



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PARt 3 – oPen InqUIRy desIgnIng yoUR InvestIgAtIons: bACteRIAl tRAnsfoRMAtIon What questions occurred to you as you performed the previous transformations? Now that you are familiar with heat shock transformation of bacterial cells, selection for transformants, and plasmid construction, design an experiment to investigate one of your questions.

Your questions may revolve around modeling how bacterial strains become antibiotic resistant, how you might use a simple pUC8 (or pUC19) plasmid vector to engineer a solution to a problem like human insulin production in a non-human system, or how other methods of transformation are more efficient than the heat shock method, etc. Remember to discuss how you will maintain safety in your experiment and any potential ethical issues that could arise in your experiment.

Before starting your experiment, plan your investigation in your lab notebook. Have your teacher check over and initial your experiment design. Once your design is approved, investigate your hypothesis. Be sure to record all observations and data in your laboratory sheet or notebook.

Use the following steps when designing your experiment.

1. Define the question or testable hypothesis.

2. Describe the background information. Include previous experiments.

3. Describe the experimental design with controls, variables, and observations.

4. Describe the possible results and how they would be interpreted.

5. List the materials and methods to be used.

6. Note potential safety issues.

After the plan is approved by your teacher:

7. The step-by-step procedure should be documented in the lab notebook. This includes recording the calculations of concentrations, etc. as well as the actual weights and volumes used.

eXPeRIMent desIgn tIPs

The College Board encourages peer review of student investigations through both formal and informal presentation with feedback and discussion. Assessment questions similar to those on the AP exam might resemble the following ques-tions, which also might arise in peer review:

Explain the purpose of a procedural step.

Identify the independent variables and the dependent variables in an experiment.

What results would you expect to see in the control group? The experimental group?

How does XXXX concept account for YYYY findings?

Describe a method to determine XXXX.

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8. The results should be recorded (including drawings, photos, data print outs).

9. The analysis of results should be recorded.

10. Draw conclusions based on how the results compared to the predictions.

11. Limitations of the conclusions should be discussed, including thoughts about improving the experimental design, statistical significance and uncontrolled variables.

12. Further study direction should be considered.

PARt 3 – oPen InqUIRy (ContInUed)eXPeRIMent desIgn tIPs

Consider the following questions when designing your experiment:

The entire experiment will be designed to provide an answer for the described problem or the question asked.

When describing the experimental group, identify the dependent and independent variable within the experimental group.

Record ALL observations and any questions generated during the experiment in your laboratory notebook or the provided laboratory sheet.

When drawing conclusions, use specific data from your experiment to support your conclusions.

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eXtensIon ACtIvItIesDivide the class into two groups. One half of the class will represent a team of doctors from a local hospital. The other half of the class will represent a team of genetic engineers from a local biotechnology company. Each team must present a justification for their beliefs about the beneficial or detrimental aspects of antibiotic-resistant organisms.

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MAteRIAl sAfety dAtA sheets




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MAteRIAl sAfety dAtA sheets (ContInUed)




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Material Safety Data Sheet Page 1 of 2

© 2008, Scholar Chemistry. All Rights Reserved. 1/23/2009

Calcium Chloride Solution 0.1MMSDS # 148.00

Section 1: Product and Company Identification

Calcium Chloride Solution 0.1MSynonyms/General Names: Calcium Chloride, Hydrated

Product Use: For educational use only

Manufacturer: Columbus Chemical Industries, Inc., Columbus, WI 53925.

24 Hour Emergency Information Telephone Numbers

CHEMTREC (USA): 800-424-9300 CANUTEC (Canada): 613-424-6666

ScholAR Chemistry; 5100 W. Henrietta Rd, Rochester, NY 14586; (866) 260-0501; www.Scholarchemistry.com

Section 2: Hazards Identification

Clear, colorless liquid; no odor. HMIS (0 to 4)

CAUTION! Body tissue irritant and slightly toxic by ingestion.

Target organs: None known

This material is considered hazardous by the OSHA Hazard Communication Standard (29 CFR 1910.1200).

Section 3: Composition / Information on Ingredients

Calcium Chloride, Anhydrous, (10035-04-8), 1-2%. Water (7732-18-5), 98-99%.

Section 4: First Aid Measures

Always seek professional medical attention after first aid measures are provided.

Eyes: Immediately flush eyes with excess water for 15 minutes, lifting lower and upper eyelids occasionally.

Skin: Immediately flush skin with excess water for 15 minutes while removing contaminated clothing.

Ingestion: Call Poison Control immediately. Rinse mouth with cold water. Give victim 1-2 cups of water or milk to drink.

Induce vomiting immediately.

Inhalation: Remove to fresh air. If not breathing, give artificial respiration.

Section 5: Fire Fighting Measures

When heated to decomposition, emits acrid fumes. 0

Protective equipment and precautions for firefighters: Use foam or dry chemical to extinguish fire. 0 0

Firefighters should wear full fire fighting turn-out gear and respiratory protection (SCBA). Cool

container with water spray. Material is not sensitive to mechanical impact or static discharge.

Section 6: Accidental Release Measures

Use personal protection recommended in Section 8. Isolate the hazard area and deny entry to unnecessary and unprotected

personnel. Contain spill with sand or absorbent material and place in sealed bag or container for disposal. Ventilate and wash

spill area after pickup is complete. See Section 13 for disposal information.

Section 7: Handling and Storage Green

Handling: Use with adequate ventilation and do not breathe dust or vapor. Avoid contact with skin, eyes, or clothing. Wash

hands thoroughly after handling.

Storage: Store in General Storage Area [Green Storage] with other items with no specific storage hazards. Store in a cool, dry,

well-ventilated, locked store room away from incompatible materials.

Section 8: Exposure Controls / Personal Protection

Use ventilation to keep airborne concentrations below exposure limits. Have approved eyewash facility, safety shower, and fire

extinguishers readily available. Wear chemical splash goggles and chemical resistant clothing such as gloves and aprons. Wash

hands thoroughly after handling material and before eating or drinking. Exposure guidelines: Calcium Chloride: OSHA PELN/A,

ACGIH: TLV: N/A, STEL: N/A.

Health 1

Fire Hazard 0

Reactivity 0




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MSDS # 148.00 Calcium Chloride Solution 0.1M Scholar Chemistry

Section 9: Physical and Chemical Properties

Molecular formula CaCl2•2H2O Appearance Clear, colorless liquid.

Molecular weight 147.02. Odor No odor.

Specific Gravity 1.00 g/mL @ 20°C Odor Threshold N/A.

Vapor Density (air=1) N/A. Solubility Soluble.

Melting Point N/A. Evaporation rate N/A (Butyl acetate = 1).

Boiling Point/Range N/A. Partition Coefficient N/A (log POW).

Vapor Pressure (20°C) N/A. pH N/A.

Flash Point: N/A. LEL N/A.

Autoignition Temp.: N/A. UEL N/A.N/A = Not available or applicable

Section 10: Stability and Reactivity

Avoid heat and moisture.

Stability: Stable under normal conditions of use and storage. Hygroscopic.

Incompatibility: Sulfuric acid, aluminum and ferrous metals.

Shelf life: Poor Shelf life, store in a cool, dry environment.

Section 11: Toxicology Information

Acute Symptoms/Signs of exposure: Eyes: Redness, tearing, itching, burning, conjunctivitis. Skin: Redness, itching.

Ingestion: Irritation and burning sensations of mouth and throat, nausea, vomiting and abdominal pain. Inhalation: Irritation of

mucous membranes, coughing, wheezing, shortness of breath,

Chronic Effects: No information found.

Sensitization: none expected

Calcium Chloride: LD50 [oral, rat]; 1000 mg/kg; LC50 [rat]; N/A; LD50 Dermal [rabbit]; N/A

Material has not been found to be a carcinogen nor produce genetic, reproductive, or developmental effects.

Section 12: Ecological Information

Ecotoxicity (aquatic and terrestrial): Not considered an environmental hazard.

Section 13: Disposal Considerations

Check with all applicable local, regional, and national laws and regulations. Local regulations may be more stringent than

regional or national regulations. Small amounts of this material may be suitable for sanitary sewer or trash disposal.

Section 14: Transport Information

DOT Shipping Name: Not regulated by DOT. Canada TDG: Not regulated by TDG.

DOT Hazard Class: Hazard Class:

Identification Number: UN Number:

Section 15: Regulatory Information

EINECS: Not listed . WHMIS Canada: D2B: Toxic Material.

TSCA: All components are listed or are exempt. California Proposition 65: Not listed.

The product has been classified in accordance with the hazard criteria of the Controlled Products Regulations and the MSDS

contains all the information required by the Controlled Products Regulations.

Section 16: Other Information

Current Issue Date: January 23, 2009Disclaimer: Scholar Chemistry and Columbus Chemical Industries, Inc., (“S&C”) believes that the information herein is factual but is not intended to be all

inclusive. The information relates only to the specific material designated and does not relate to its use in combination with other materials or its use as to any

particular process. Because safety standards and regulations are subject to change and because S&C has no continuing control over the material, those

handling, storing or using the material should satisfy themselves that they have current information regarding the particular way the material is handled, stored

or used and that the same is done in accordance with federal, state and local law. S&C makes no warranty, expressed or implied, including (without

limitation) warranties with respect to the completeness or continuing accuracy of the information contained herein or with respect to fitness for any

particular use.



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CMSICulture Media & Supplies, Inc. Luria Agar Prepared

118 D Kirkland Circle Catalog #: CM213-Oswego, Illinois 60543-8069P: 630-499-5000 • F: 630-499-9900

www.culturemediasupplies.com

Hazards Identification

Hazard DescriptionXi IrritantDangerous ComponentsSodium Chloride: 25.0%Information pertaining to particular dangers for man and environmentR 36/37/38 Irritating to eyes, respiratory system and skin.Classification SystemThe classification was made according to the latest editions of international substanceslists, and expanded upon from company and literature data.NFPA ratings (scale 0-4)Health = 1Fire = 0Reactivity = 0First Aid Measures

General information: No Special measures required.After inhalation: Seek fresh air; consult doctor in case of complaints.After skin contact: Immediately wash with water and soap and rinse thoroughly.After eye contact: Rinse opened eye for several minutes under running water. Ifsymptoms persist, consult a doctor.After swallowing: Call a doctor immediately.Information for doctor: Show this sheet.Toxicological Information

Acute toxicity:LD/Lc50 values that are relevant for classification:7647-14-5 sodium chloride. Oral: LD50: 3.000 mg/kg (rat)Primary irritant effect:on the skin: Irritant to skin and mucous membranes.on the eye: Irritating effect.Sensitization: No sensitizing effects knownSubacute to chronic toxicity:Target organs: respiratory tract, eyes, and skin.Additional toxicological information:When used and handled according to specifications, the product does not have anyharmful effects according to our experience and the information provided to us.

Material Safety Data Sheet

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CMSICulture Media & Supplies, Inc. Luria Agar Prepared



Handling and Storage

HandlingInformation for safe handling: Prevent formation of dust.Information about protection against explosives and fires:No special measures required.StorageRequirements to be met by storerooms and receptacles: <30 CInformation about storing conditions:Sore in a cool, dry condition in well sealed receptacle.Class according to regulations on flammable liquids: Void

Exposure Controls and Personal Protection

Components with limit values that require monitoring at the workplace:This product does not contain any relevant quantities of materials with critical valuesthat have to be monitored at the workplace.Personal protective equipmentGeneral protective and hygienic measures:The usual precautionary measures for handling chemicals should be followed.Breathing equipment: Not required.Protection of hands: Protective gloves.Eye protection: Safety glasses.Body protection: Protective work clothing (lab coat)



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CMSICulture Media & Supplies, Inc. Tryptic Soy Broth Prepared



Hazards Identification

Hazard DescriptionXi IrritantDangerous ComponentsSodium Chloride: 12.5%Information pertaining to particular dangers for man and environmentR 36/37/38 Irritating to eyes, respiratory system and skin.Classification SystemThe classification was made according to the latest editions of international substanceslists, and expanded upon from company and literature data.NFPA ratings (scale 0-4)Health = 1Fire = 0Reactivity = 0First Aid Measures

General information: No Special measures required.After inhalation: Seek fresh air; consult doctor in case of complaints.After skin contact: Immediately wash with water and soap and rinse thoroughly.After eye contact: Rinse opened eye for several minutes under running water. Ifsymptoms persist, consult a doctor.After swallowing: Call a doctor immediately.Information for doctor: Show this sheet.Toxicological Information

Acute toxicity:LD/Lc50 values that are relevant for classification:7647-14-5 sodium chlorideOral: LD50: 3.000 mg/kg (rat)Primary irritant effect:on the skin: Irritant to skin and mucous membranes.on the eye: Irritating effect.Sensitization: No sensitizing effects knownSubacute to chronic toxicity:Target organs: respiratory tract, eyes, and skin.Additional toxicological information:When used and handled according to specifications, the product does not have anyharmful effects according to our experience and the information provided to us.


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CMSICulture Media & Supplies, Inc. Tryptic Soy Broth Prepared



Handling and Storage

HandlingInformation for safe handling: Prevent formation of dust.Information about protection against explosives and fires:No special measures required.StorageRequirements to be met by storerooms and receptacles: <30 CInformation about storing conditions:Store in a cool, dry condition in well sealed receptacle.Class according to regulations on flammable liquids: Void

Exposure Controls and Personal Protection

Components with limit values that require monitoring at the workplace:This product does not contain any relevant quantities of materials with critical valuesthat have to be monitored at the workplace.Personal protective equipmentGeneral protective and hygienic measures:The usual precautionary measures for handling chemicals should be followed.Breathing equipment: Not required.Protection of hands: Protective gloves.Eye protection: Safety glasses.Body protection: Protective work clothing (lab coat)

Data Sheet


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Water, DeionizedMSDS # 786.70

Section 1: Product and Company Identification

W ater , D eionizedSynonyms/ G ener al N ames: DI water

P roduct Use: For educational use only

M anufactu rer : Columbus Chemical Industries, Inc., Columbus, WI 53925.

24 H ou r E mergency Infor mation T elephone N umbersC H E M T R E C (USA ): 800-424-9300 C A N U T E C ( C anada): 613-424-6666

ScholAR Chemistry; 5100 W. Henrietta Rd, Rochester, NY 14586; (866) 260-0501; www.Scholarchemistry.com

Section 2: Hazards Identification

Clear, colorless liquid H M IS (0 to 4)

This material is not considered hazardous. Not for human consumption .

Target organs: None known

This material is not considered hazardous by the OSHA Hazard Communication Standard (29 CFR 1910.1200).

Section 3: Composition / Information on Ingredients

Water (7732-18-5), 100%.

Section 4: First Aid Measures

Always seek professional medical attention after first aid measures are provided.Do not d r in k.

U ncontaminated water should not pose any ill health effects. If ill effects devlop, seek p rofessional medical attentionimmediately.

Section 5: Fire Fighting Measures

Avoid contact with Dangerous When Wet materials and chemicals along with water reactive materials. 0. 0 0

Section 6: Accidental Release Measures

Use personal protection recommended in Section 8. Isolate the hazard area and deny entry to unnecessary and unprotected

personnel. Contain spill with sand or absorbent material and place in sealed bag or container for disposal. Wash and dry spill

area after pickup is complete. See Section 13 for disposal information.

Section 7: Handling and Storage Green

H andling: Wash hands thoroughly after handling.

Stor age: Store in General Storage Area [Green Storage] with other items with no specific storage hazards. Store in a cool, dry,

well-ventilated, locked store room away from incompatible materials.

Section 8: Exposure Controls / Personal Protection

Use ventilation to keep airborne concentrations below exposure limits. Have approved eyewash facility, safety shower, and fire

extinguishers readily available. Wear chemical splash goggles and chemical resistant clothing such as gloves and aprons. Wash

hands thoroughly after handling material and before eating or drinking. Use NIOSH-approved respirator with a dust cartridge.

Exposure guidelines: DI water: OSHA PEL: N/A; ACGIH: TLV: N/A; STEL: N/A.

H ealth 0F i re H aza r d 0Reactivity 0


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